In recent years, attempts have been made to modify virus genomes by using genetic engineering techniques to make viruses which selectively replicate in cancer cells and apply these viruses to cancer treatment.
The concept of applying recombinant viruses to cancer treatment was advocated by Martuza et al. in 1991 (refer to, for example, Non-patent Document 1). Since many viruses are themselves pathogenic, direct administration to humans and so forth has detrimental effects on normal cells as well. However, by deleting or mutating specific genes by using genetic engineering, viruses can be made which are incapable of replicating in normal cells but are capable of replicating in actively growing tumor cells through mechanisms such as compensation of the deleted gene function.
Recombinant oncolytic viruses are genetically modified so as to selectively replicate only in cancer cells, replicate in situ upon infecting cancer cells and directly destruct host cancer cells during that process. Subsequently, the progeny viruses scatter around and infect cancer cells again. Then, they repeat replication, cell killing and infection and thus, produce anti-tumor effects. On the other hand, therapeutic viruses which have infected normal cells cannot replicate so that they do not cause damage to the normal tissue.
Examples of such mutant viruses which have been developed thus far include a mutant virus obtained by deleting the thymidine kinase (tk) gene from the genome of herpes simplex virus type 1 (HSV-1) (refer to, for example, the above-mentioned Non-patent Document 1); HSV-1 (which will hereinafter be called “G207”) obtained by deleting both two copies of the γ34.5 gene and inactivating the ICP6 gene (refer to, for example, Non-patent Documents 2 to 14); and HSV-1 (which will hereinafter be called “G47Δ”, refer to, for example, Patent Document 1 and Non-patent Document 15) obtained by deleting the ICP47 gene (also called α47 gene) in addition to deletion of both two copies of the γ34.5 gene and inactivation of the ICP6 gene. Although these mutant viruses are unable to replicate in normal cells, they retain the replication ability in tumor cells.
In particular, as a result of mutation of three genes, the G47Δ developed by the present inventors has high tumor specificity in virus replication and has high cytocidal effects limited to tumor cells, while it shows no toxicity in the normal tissue so that its safety is not damaged and antitumor effects are markedly improved compared with the parent G207 virus. In addition, the G47Δ permits production of a virus formulation having a higher titer compared with G207 so that G47Δ is expected to have a higher therapeutic effect when their volumes are equal.
FIG. 11 shows the structure of G47Δ. As shown in this figure, G47Δ has a 1-kb deletion in two γ34.5 genes and a 312-b depletion in the α47 gene and in addition, its ICP6 gene has been inactivated by inserting the lacZ-gene therein.
The γ34.5 gene is a gene related to pathogenicity of HSV-1 and a mutant obtained by deleting this gene has markedly reduced virus replication ability in normal cells. When virus infection occurs in normal cells, double-stranded RNA-activated protein kinase (PKR) is phosphorylated and the resulting protein kinase phosphorylates a translation initiation factor elF-2a, resulting in blocking of protein synthesis in cells containing a viral protein. Although the γ34.5 gene product antagonizes the phosphorylated PKR and permits synthesis of a viral protein, the γ34.5 gene-deficient HSV-1 cannot replicate in normal cells. It is however presumed that in tumor cells, the universally low phosphorylation level of PKR enables even the γ34.5 gene-deficient HSV-1 to replicate.
The ICP6 gene is a gene encoding the large subunit of ribonucleotide reductase (RR). RR is an enzyme necessary for virus DNA synthesis. Inactivation of this gene prevents virus replication in non-dividing cells. Only in cells which divide actively and have increased RR activity, the deficiency of the RR activity due to deletion of the ICP6 gene is compensated, enabling virus replication.
The α47 gene product inhibits the transporter associated with antigen presentation (TAP) in host cells to control expression of the MHC class I on the cell surface, thereby having an effect of suppressing presentation of a viral protein and escaping from immunosurveillance of the host. Accordingly, it is expected that in the α47-gene-deleted HSV-1, expression of the MHC class I of host cells is maintained and a stimulus to antitumor immune cells increases.
In addition, G47Δ also has a deletion of the promoter of the US11 gene which overlaps with the α47 gene so that the timing of US11 gene expression is accelerated. As a result, this gene functions as a second site suppressor of the γ34.5 gene mutation and it restores, only in tumor cells, the reduced virus replication ability in the γ34.5-deficient HSV-1.
As described above, G47Δ has four artificial mutations separated from each other on the virus genome so that there is almost no possibility of it reverting to wild type HSV-1. This suggests that G47Δ is a therapeutic virus having high tumor specificity and high safety and excellent in versatility and usefulness.
In G47Δ, however, the RR activity necessary for replication is compensated by the RR activity of tumor cells so that in theory, virus replication is not conducted efficiently in tumor cells not having sufficiently increased RR activity and there is a possibility of failing to obtain sufficient antitumor effects.